Aliso: A Journal of Systematic and Evolutionary Botany

Volume 34 | Issue 1 Article 3

2016 A Morphometric Analysis of campylopodum and Arceuthobium divaricatum (Viscaceae) Robert L. Mathiasen Northern Arizona University, Flagstaff

Shawn C. Kenaley Cornell University, Ithaca

Carolyn M. Daugherty Northern Arizona University, Flagstaff

Follow this and additional works at: http://scholarship.claremont.edu/aliso Part of the Botany Commons

Recommended Citation Mathiasen, Robert L.; Kenaley, Shawn C.; and Daugherty, Carolyn M. (2016) "A Morphometric Analysis of Arceuthobium campylopodum and Arceuthobium divaricatum (Viscaceae)," Aliso: A Journal of Systematic and Evolutionary Botany: Vol. 34: Iss. 1, Article 3. Available at: http://scholarship.claremont.edu/aliso/vol34/iss1/3 Aliso, 34(1), pp. 9–23 ISSN 0065-6275 (print), 2327-2929 (online)

A MORPHOMETRIC ANALYSIS OF ARCEUTHOBIUM CAMPYLOPODUM AND ARCEUTHOBIUM DIVARICATUM (VISCACEAE)

ROBERT L. MATHIASEN1,4,SHAWN C. KENALEY2, AND CAROLYN M. DAUGHERTY3

1School of Forestry, Northern Arizona University, Flagstaff, Arizona 86011; 2School of Integrative Science, Plant Pathology, and Plant-Microbe Biology Section, Cornell University, Ithaca, New York 14853; 3Department of Geography, Planning, and Recreation, Northern Arizona University, Flagstaff, Arizona 86011 4Corresponding author ([email protected])

ABSTRACT Although the classification of pinyon dwarf mistletoe (Arceuthobium divaricatum, Viscaceae) has not been controversial to any extent since Engelmann described it in 1878, a recent taxonomic treatment has included this species in western dwarf mistletoe (A. campylopodum). While pinyon dwarf mistletoe is only known to parasitize pinyon (Pinus subsection Cembroides), western dwarf mistletoe as it has been known since the late 1800s is a principal parasite of Pinus ponderosa and P. jeffreyi and has never been observed parasitizing pinyon pines. With reservations about the recent classification of pinyon dwarf mistletoe and its treatment under A. campylopodum, we undertook this study to examine in detail the morphological characteristics of pinyon dwarf mistletoe and compare them with those of western dwarf mistletoe. Pinyon and western dwarf mistletoe populations were sampled throughout most of their geographic ranges and morphological traits including plant, flower, fruit, and seed dimensions were measured. Thereafter, we compared morphological characteristics between A. campylopodum and A. divaricatum using univariate and multivariate statistics to determine significant differences among morphologies of both male and female . Our analyses clearly demonstrated that pinyon and western dwarf mistletoe are morphologically distinct as originally proposed by G. Engelmann in the late 19th century. Furthermore, the host affinities of the two taxa clearly distinguish them from each other. Therefore, we recommend that A. campylopodum and A. divaricatum continue to be classified as separate species. Morphological differences between these species are summarized and a key is provided for use in their field identification. Key words: Arceuthobium, dwarf mistletoes, morphological characters, multivariate analyses, Pinus

INTRODUCTION Zucc. (Mexican pinyon), P. discolor D.K. Bailey & Hawksw. (border pinyon), P. californiarum D.K. Bailey (California The dwarf mistletoes (Arceuthobium spp., Viscaceae) are singleleaf pinyon), and P. californiarum subsp. fallax (Little) among the most damaging parasites of commercially valuable D.K. Bailey (Arizona singleleaf pinyon). However, pinyon conifers in the western United States and Canada (Hawksworth dwarf mistletoe only parasitizes these latter five pinyons in et al. 2002). Severely infected trees suffer reduced growth and a few locations (Hawksworth and Wiens 1996). Major premature mortality and are often predisposed to attack by differences in the host affinities of these dwarf mistletoes based insects. Two widespread and abundant dwarf mistletoes found on field observations has led to the claim that P. jeffreyi is in the western United States are A. campylopodum Engelm. immune to infection by A. divaricatum and that P. monophylla (western dwarf mistletoe) and A. divaricatum Engelm. (pinyon is immune to infection by A. campylopodum (Hawksworth and dwarf mistletoe) (Hawksworth and Wiens 1972, 1996). Western Wiens 1996). dwarf mistletoe parasitizes several species of pines including Arceuthobium has long been considered a taxonomically Pinus ponderosa Douglas ex Lawson & C. Lawson (ponderosa difficult genus because of the morphological and phenological ), P. jeffreyi Grev. & Balf. (Jeffrey pine), P. coulteri D. Don similarities among taxa (Gill 1935; Hawksworth and Wiens (Coulter pine), and P. attenuata Lemmon (knobcone pine), but 1972, 1996; Hawksworth et al. 2002). Morphological reduction has never been reported parasitizing pinyon pines (Pinus and similarity and sexual dimorphism hamper identification of subsection Cembroides) present in the West (Hawksworth and Arceuthobium taxa in the field and have resulted in major Wiens 1972, 1996). In contrast, pinyon dwarf mistletoe differences in taxonomic treatments. However, little disagree- exclusively parasitizes pinyons, primarily P. edulis Engelm. ment regarding the classification of A. divaricatum at the P monophylla (Colorado pinyon) and . Torrey & Fre´m. specific level has been presented in the literature since the late (singleleaf pinyon). It has also been reported to infect P. 1800s when it was first described by George Engelmann quadrifolia P cembroides Parlat. ex Sudw. (Parry pinyon), . (Engelmann 1878). In the first monograph of Arceuthobium in the United States, Gill (1935) classified A. divaricatum as ’ 2016, The Author(s), CC-BY. This open access article is a host form of A. campylopodum that exclusively parasitized distributed under a Creative Commons Attribution License, which pinyons and hence, under this system, any dwarf mistletoe allows unrestricted use, distribution, and reproduction in any medium, found on a pinyon was classified as A. campylopodum provided that the original author(s) and source are credited. Articles (Engelm.) forma divaricatum (Engelm.) Gill. The host-form can be downloaded at http://scholarship.claremont.edu/aliso/. system proposed by Gill worked well for pinyon dwarf 10 Mathiasen, Kenaley, and Daugherty ALISO mistletoe because no other dwarf mistletoes in the United States infect pinyons. However, this system did not work well in high elevation mixed conifer forests where other host forms of A. campylopodum were sympatric and, to varying degrees, parasitized more than one host. For example, Gill’s host-form system classified all dwarf mistletoes parasitizing a true fir (Abies Mill.) as A. campylopodum Engelm. forma abietinum (Engelm.) Gill, even if the dwarf mistletoe also parasitized hemlock (Tsuga [Endl.] Carrie`re) in the same locality. Gill’s treatment required that the mistletoe on hemlock be classified as A. campylopodum Engelm. forma tsugensis (Rosendahl) Gill, even though the mistletoe on the true fir and the mistletoe on the hemlock were morphologically identical; they were the same mistletoe. Gill’s host-form system lacked the recognition that a dwarf mistletoe could cross-infect more than one host species at the same location, which resulted in classifying the same dwarf mistletoe as different forma due solely to the fact that it was on a different tree species. The inadequacies of Gill’s (1935) classification system soon became obvious, and in their first monograph of Arceutho- bium, Hawksworth and Wiens (1972) classified all of Gill’s host forms of A. campylopodum as species, including A. divaricatum, based on differences in morphology, phenology, chemistry, and host specificity. In their revised monograph for Arceuthobium, Hawksworth and Wiens (1996) maintained A. divaricatum as a separate species from A. campylopodum, based on their distinctive morphologies and host specificities. Maintaining A. divaricatum and A. campylopodum as separate species was further supported by isozyme (Nickrent 1986) and molecular analyses using nrDNA ITS and chloroplast trn-L Fig. 1. Approximate locations of collection sites for Arceuthobium sequences (Nickrent et al. 1994, 2004). campylopodum in California, Idaho, Nevada, Oregon, and Washing- Despite the seemingly overwhelming evidence in the ton. Filled circles represent locations where plants were collected from literature related to the classification of Arceuthobium divar- Pinus ponderosa. Open circles represent locations where plants were icatum as a separate species from A. campylopodum, Kuijt collected from P. jeffreyi. Numbers correspond to locations in included A. divaricatum under A. campylopodum in the revised Appendix 1 (map reproduced with permission by the California Bo- tanical Society (Madron˜o 62: 6). Jepson Manual (Baldwin et al. 2012). The use of the revised Jepson Manual by many natural scientists in California has MATERIALS AND METHODS resulted in at least three problems: (1) populations of A. divaricatum are being identified as A. campylopodum, (2) Morphological Measurements herbarium specimens of A. divaricatum are being annotated as Morphological data for A. campylopodum from 60 pop- representing A. campylopodum, and (3) populations of A. ulations, 30 each from Pinus ponderosa and P. jeffreyi, divaricatum are appearing on maps as locations for A. (Mathiasen and Kenaley 2015a; Fig. 1 and Appendix 1) was campylopodum. An example of this third problem can be seen augmented by morphological data collected for A. divaricatum in the CALFLORA map for A. campylopodum (http://www. from 60 populations distributed through most of its geo- calflora.org/). By classifying A. divaricatum and 10 other taxa graphic range in 2014–2015 (Fig. 2 and Appendix 2). Most of recognized by Hawksworth and Wiens (1996) as separate these populations were from locations where A. divaricatum species under only one species, A. campylopodum, the utility of was parasitizing (30 populations), but we also such maps for locating dwarf mistletoe populations by sampled 23 populations of A. divaricatum on P. monophylla, botanists and foresters working in California is greatly and seven populations on P. californiarum subsp. fallax diminished (Mathiasen and Kenaley 2015a). (Fig. 2). Because we were only able to sample one population Because of the confusion created by the recent inclusion of of A. divaricatum parasitizing P. californiarum subsp. califor- Arceuthobium divaricatum under A. campylopodum, we un- niarum in southern California, one population parasitizing P. dertook this study to further compare the morphological cembroides in western Texas, and two populations of A. characteristics of these dwarf mistletoes. We sampled male and divaricatum on P. discolor in New Mexico, we have not female plants, flowers, fruits, and seeds for these dwarf included morphological measurements for plants from those mistletoes on their respective pine hosts throughout most of hosts in our results. Voucher specimens for A. campylopodum their geographic ranges. We then applied both univariate and and A. divaricatum consisting of the mistletoe with host multivariate statistical analyses to compare the morphological material were deposited at the University of Arizona characteristics of plants, flowers, fruits, and seeds from the Herbarium, Tucson (ARIZ), the Herbarium of Rancho Santa sampled populations. Ana Botanical Garden, Claremont, CA (RSA), or the Deaver VOLUME 34(1) Morphometric Analysis of Dwarf Mistletoes 11

Fig. 2. Approximate locations of collection sites for Arceuthobium divaricatum in Arizona, California, Colorado, Nevada, New Mexico, and Utah. Filled circles represent locations where plants were collected from . Open circles represent locations where plants were collected from P. californiarum subsp. fallax. Filled squares represent locations where plants were collected from P. edulis. Numbers correspond to locations in Appendix 2.

Herbarium, Northern Arizona University, Flagstaff (ASC). base of plants was included in our morphological analyses Voucher and specific population data, including collection because these characters have been frequently reported for dwarf numbers, collection dates, and GPS coordinates, have been mistletoes and provide information on the relative size and archived electronically in SEINet (Southwest Environmental thickness of male and female plants (Hawksworth and Wiens Information Network: (http://swbiodiversity.org/portal/index. 1972, 1996; Mathiasen and Daugherty 2007, 2009a, 2009b, 2013; php) or the Consortium of California Herbaria (http://ucjeps. Mathiasen and Kenaley 2015a,2015b). The length of the third berkeley.edu/consortium). internode was determined by measuring from the top of For each mistletoe population, 10 male and 10 female the second internode above the base of a plant to the top of the plants were randomly collected and the dominant plant third internode, locations which are easily observed (see Fig. 2.1 (largest plant) of each sex was used for morphological and 2.3 in Hawksworth and Wiens 1996). The width of the measurements. The dwarf mistletoe plant characters mea- third internode was measured at its midpoint. Staminate spike sured were those used by Hawksworth and Wiens (1996) for and flower measurements were made during the peak of anthesis. their taxonomic classification of Arceuthobium.Thesein- Likewise, fruit and seed measurements were made during the cluded: (1) height, basal diameter, third internode length peak of seed dispersal. and width, and color of male and female plants; (2) mature fruit length, width, and color from female plants; (3) seed Statistical Analyses length, width and color; (4) length and width of mature staminate spikes; (5) staminate flower diameters for 3- and We assessed whether mean values for morphological 4-merous flowers (4-merous flowers were rarely observed for characters differed significantly between the two dwarf A. divaricatum); (6) length and width of staminate flower mistletoes and between comparable characters from plants petals; and (7) anther diameter and anther distance from the parasitizing Pinus edulis, P. monophylla, and P. californiarum petal tip. subsp. fallax using one-way analysis of variance (ANOVA) Plants typically were measured within 12 h, but no later than and, when appropriate, a posthoc Tukey’s Honestly Signifi- 24 h after collection. Only plants attached to their host’s branch cant Difference (HSD) Test (a 5 0.05) (Zimmerman 2004). and fully turgid were measured. Quantitative measurements were Quadratic discriminant function analyses (DFA) were also made using a digital caliper (Mitutoyo America Corp., Aurora, performed separately to assess whether female or male plants IL) and a 7X hand lens equipped with a micrometer (Bausch & of A. campylopodum and A. divaricatum can be delimited to Lomb, Bridgewater, NJ). The basal diameter of plants was species according to morphological characters (predicted measured at the point where the plant was attached to the host versus actual; Quinn and Keough 2002; Fig. 3). Discriminant branch. The width and length of the third internode above the function analyses for female and male plants were conducted 12 Mathiasen, Kenaley, and Daugherty ALISO

Fig. 3. Canonical plots for discriminant function analyses (DFA) of Arceuthobium campylopodum and, according to host, A. divaricatum based on morphological characteristics of female (A, C) and male plants (B, D). Multivariate means (cross-hairs) were calculated using complete data for each species by sex (A, B), whereas, to further validate the DFA, means were also calculated using resampled data (25 complete records/ species) of female (C) and male plants (D), respectively. For each species (A–D), the inner ellipse is a 95% confidence limit for the mean, and the outer ellipse is a normal contour where approximately 50% of plants for each species reside. Correct classification percentages for male and female plants by DFA (complete and resampled) are presented in Table 6. on two separate datasets: (1) complete records for A. prior probabilities for each host-dwarf mistletoe combination campylopodum as well as A. divaricatum partitioned by host (25%, partitioned dataset) or species (50%, combined dataset) (i.e., P. edulis, P. monophylla, and P. californiarum subsp. rather than proportional to their actual host and/or species fallax), and (2) complete records for A. campylopodum and, membership. Discriminant function analyses were parameter- collectively, A. divaricatum across all three hosts combined. ized to include equal prior probabilities in order to remove Actual host or species membership was defined a priori via experimental bias (i.e., a priori identification) from the posthoc field diagnosis and, although previous molecular analyses classification (%, predicted/actual) of dwarf mistletoes by host- supported the separation of A. campylopodum and A. dwarf mistletoe combination and species membership. For divaricatum at the specific level (Nickrent 1986; Nickrent comparisons of species membership, standardized correlation et al. 2004), female and male DFAs were executed using equal coefficients for morphological characters were also calculated VOLUME 34(1) Morphometric Analysis of Dwarf Mistletoes 13

Table 1. Morphological measurements for Arceuthobium campylopodum collected from Pinus jeffreyi and P. ponderosa and for A. divaricatum collected from P. edulis, P. monophylla, and P. californiarum subsp. fallax. Data are listed as mean, (SD), range, [n]. Means followed by different capital letters in the same row were significantly different using ANOVA (a 5 0.05). Lower case letters in brackets designate sample sizes already listed in the same column. Plant heights are in cm and all other measurements in mm.

Character Arceuthobium campylopodum Arceuthobium divaricatum

Plant height Female 10.4 A (2.7) 3.9225.4 [600a] 10.9 B (2.7) 5.2222.7 [600a] Male 9.7 A (3.0) 3.6221.6 [a] 11.7 B (3.3) 5.7230.4 [a] Basal diameter Female 3.4 A (0.7) 1.726.9 [a] 2.6 B (0.5) 1.325.0 [a] Male 3.2 A (0.6) 1.826.8 [a] 2.6 B (0.5) 1.624.9 [a] Length of third internode Female 13.0 A (3.1) 5.7229.3 [a] 11.6 B (2.6) 5.8221.9 [a] Male 12.0 A (3.3) 4.2223.2 [a] 11.7 A (2.6) 5.5221.8 [a] Width of third internode Female 2.5 A (0.4) 1.622.7 [a] 1.9 B (0.3) 1.223.2 [a] Male 2.5 A (0.4) 1.422.6 [a] 1.9 B (0.3) 1.223.1 [a] Staminate spike length 12.7 A (4.8) 3.7241.0 [760b] 9.5 B (3.1) 3.2231.8 [580b] Staminate spike width 3.0 A (0.3) 2.324.2 [b] 1.7 B (0.1) 1.122.3 [b] Flower diameter 3-merous 3.1 A (0.4) 3.124.5 [400] 2.2 B (0.3) 1.423.2 [b] 4-merous 4.2 A (0.5) 3.026.2 [360] 3.1 B (0.3) 2.224.1 [50] Petal lobe length 1.6 A (0.2) 0.922.4 [b] 1.1 B (0.1) 0.721.6 [b] Petal lobe width 1.4 A (0.2) 0.722.4 [b] 1.0 B (0.1) 0.621.5 [b] Anther diameter 0.6 A (0.1) 0.421.2 [b] 0.4 B (0.1) 0.320.7 [b] Anther distance from tip 0.6 A (0.1) 0.221.1 [b] 0.4 B (0.1) 0.220.8 [b] Fruit length 5.4 A (0.5) 4.027.2 [480c] 4.4 B (0.3) 3.225.3 [590c] Fruit width 3.7 A (0.4) 2.625.6 [c] 2.6 B (0.2) 1.923.5 [c] Seed length 3.5 A (0.4) 2.324.7 [c] 2.2 B (0.3) 1.623.1 [c] Seed width 1.5 A (0.2) 1.022.0 [c] 1.1 B (0.1) 0.821.3 [c] to determine the overall contribution of each character to the The staminate spikes of Arceuthobium campylopodum were discriminant function. Likewise, when appropriate, stepwise significantly longer on average and the width of staminate DFA was utilized to examine systematically the smallest spikes was significantly wider than for A. divaricatum; the number of morphological characteristics, female or male, mean width of staminate spikes of A. divaricatum being nearly resulting in the highest percentage in correct classification (%, half as wide as those of A. campylopodum. A major difference predicted/actual). To further validate the DFAs, we resampled between A. campylopodum and A. divaricatum was that the separately the partitioned and collective data for female and latter species predominantly produced 3-merous staminate male plants; selecting at random 25 complete records per host- flowers whose mean diameter (2.2 mm) was significantly dwarf mistletoe combination or species and re-executed the smaller than that of the 3-merous flowers of A. campylopodum DFA to include all female or male morphological characters (3.1 mm; Table 1). The mean diameter of 4-merous flowers simultaneously (full-model) with each plant receiving equal was also significantly smaller for A. divaricatum, but 4-merous prior probabilities. Analysis of variance tests and DFAs were flowers were only occasionally observed and measured for computed in JMP Pro 12 (SAS Institute, Cary, North populations of A. divaricatum; only eight populations were Carolina). observed with a few 4-merous flowers. The smaller size of the staminate flowers of A. divaricatum was a result of its RESULTS significantly smaller mean petal length and width. Mean anther diameter (0.4 mm) and the distance of anthers from the Morphological Differences tips of petals also were both significantly smaller for A. The mean heights of female and male plants of Arceutho- divaricatum than A. campylopodum (Table 1). Similarly, mean bium campylopodum were significantly smaller than those of A. fruit length was much larger for A. campylopodum (5.4 mm) divaricatum (Table 1). However, even though the mean heights than for A. divaricatum (4.4 mm), as was the mean width of of male plants varied by 2 cm, the mean height of female plants fruits. Mean seed length and width were also significantly only varied by 0.5 cm. The mean length of the third internode different between the two species (Table 1). of female plants was significantly different between taxa, but Plant color is not usually an informative character for the mean length of the third internode of male plants was not distinguishing between dwarf mistletoes. However, the color of significantly different. Male and female plants of A. divar- plants of A. divaricatum was distinctly different from those of icatum were more slender than those of A. campylopodum with A. campylopodum, the former being consistently brown or dark both the mean basal diameter and mean width of the third brown to green-brown compared to the latter which ranged internode for both sexes being significantly smaller for A. from yellow, yellow-brown, or light brown (Fig. 4–5). Some- divaricatum (Table 1). times male plants of A. divaricatum were red-brown, yellow- 14 Mathiasen, Kenaley, and Daugherty ALISO

Fig. 4–5. Color variation between Arceuthobium campylopodum and A. divaricatum.—4. Female plants of A. campylopodum with nearly mature fruits in July. Note that plants are yellow-brown.—5. Female plants of A. divaricatum with nearly mature fruits in August. Note that plants are brown-green. VOLUME 34(1) Morphometric Analysis of Dwarf Mistletoes 15

Table 2. Morphological measurements for Arceuthobium divaricatum from Pinus edulis, P. monophylla, and P. californiarum subsp. fallax. Data are listed as mean, (SD) [n]. Means followed by different capital letters in the same row were significantly different using ANOVA followed by a Tukey’s Post Hoc HSD test (a 5 0.05). Lower case letters in brackets designate sample sizes already listed in the same column. Plant heights are in cm and all other measurements in mm.

Character Pinus edulis Pinus monophylla Pinus californiarum subsp. fallax

Plant height Female 10.1 A (2.2) [300a] 11.8 B (2.9) [230a] 11.5 B (2.9) [70a] Male 10.6 A (2.3) [a] 13.0 B (3.8) [a] 12.2 B (3.6) [a] Basal diameter Female 2.6 A (0.5) [a] 2.6 A (0.5) [a] 2.6 A (0.4) [a] Male 2.5 A (0.5) [a] 2.4 A (0.5) [a] 2.4 A (0.5) [a] Length of third internode Female 11.3 A (2.5) [a] 11.9 B (2.6) [a] 11.6 AB (2.8) [a] Male 11.5 A (2.4) [a] 12.0 A (2.7) [a] 11.4 A (2.6) [a] Width of third internode Female 1.9 A (0.3) [a] 1.9 A (0.3) [a] 2.0 B (0.3) [a] Male 1.9 A (0.3) [a] 2.0 A (0.3) [a] 1.9 A (0.3) [a] Staminate spike length 9.5 A (3.3) [280b] 9.3 A (2.8) [a] 9.7 A (3.3) [a] Staminate spike width 1.7 A (0.1) [b] 1.7 A (0.1) [a] 1.7 A (0.1) [a] Flower diameter 3-merous 2.2 A (0.2) [b] 2.2 A (0.2) [a] 2.2 A (0.2) [a] Petal lobe length 1.1 A (0.2) [b] 1.0 A (0.2) [a] 1.1 A (0.2) [a] Petal lobe width 1.0 A (0.1) [b] 1.0 A (0.1) [a] 1.0 A (0.1) [a] Anther diameter 0.4 A (0.1) [b] 0.4 A (0.1) [a] 0.4 A (0.1) [a] Anther distance from tip 0.4 A (0.1) [b] 0.3 B (0.1) [a] 0.4 A (0.1) [a] Fruit length 4.4 A (0.4) [290c] 4.3 A (0.3) [a] 4.4 A (0.3) [a] Fruit width 2.6 A (0.3) [c] 2.6 B (0.2) [a] 2.7 A (0.2) [a] Seed length 2.1 A (0.2) [c] 2.2 B (0.3) [a] 2.3 C (0.3) [a] Seed width 1.1 A (0.1) [c] 1.1 A (0.1) [a] 1.0 A (0.1) [a] brown or even yellow-green; very few female plants of A. Although Arceuthobium campylopodum and A. divaricatum divaricatum were these colors. have been reported to flower at approximately the same time Because Arceuthobium divaricatum parasitizes several differ- (late August to late September), we observed that A. ent pinyons, we compared the morphological characteristics of divaricatum started flowering in early August, at least two male and female plants from three of its pinyon hosts weeks earlier than A. campylopodum. Flowering periods of A. (Table 2). The heights of male and female plants collected campylopodum and A. divaricatum did overlap in mid to late from Pinus monophylla and P. californiarum subsp. fallax were August; however, these dwarf mistletoes were not found in the significantly larger than those collected from P. edulis. same stands and the difference in the start of flowering we Although female and male plants collected from P. califor- observed in California may have been related to the lower niarum subsp. fallax were shorter on average than those elevations where we made our observations for A. divaricatum. collected from P. monophylla, the means were not significantly Seed dispersal of these species occurs from late August to late different. No other significant differences were detected among September, but some populations of A. divaricatum at the the morphological characters measured for plants, flowers, or lowest elevations of its geographic range disperse seed into late fruits from the different pinyon hosts. The mean seed length October; elevation and its influence on climate may be the found in the three pinyon hosts was significantly different but primary reason why A. divaricatum disperses seed later in the only by 0.1 mm, and the mean width of seeds was not fall than A. campylopodum in California. significantly different (Table 2). Because the mean heights of male and female plants collected from P. monophylla and P. Discriminant Function Analyses californiarum subsp. fallax were not significantly different, we combined morphological measurements for plants, flowers, Because some variation in male and female morphologies in fruits, and seeds from those hosts and compared them to Arceuthobium divaricatum was evident among the three pinyon measurements for these characters from only P. edulis hosts, DFA was conducted first on separate female and male (Table 3). Again, only the mean heights of female and male plant datasets consisting of measurements of morphological plants and mean seed lengths were significantly different. We characters from A. campylopodum as well as A. divaricatum then compared characters measured from P. edulis and pooled partitioned according to host. Results from these analyses measurements from P. monophylla and P. californiarum subsp. indicated significant differences for the eight female morpho- fallax with those of A. campylopodum (Table 4). The mean logical characters of A. campylopodum and, partitioned by heights of female plants from P. edulis were not significantly host, A. divaricatum (Wilks’ l 5 0.1201, Approx. F24,3043 5 different to those of A. campylopodum, but the mean values for 136.48, P , 0.0001; Pillai’s Trace 5 0.9702, Approx. F24,3153 5 all other characters (except third internode length of male 62.80, P , 0.0001). Likewise, significant differences were also plants) of A. divaricatum were significantly different from found among the 10 male plant characteristics examined for A. those of A. campylopodum. divaricatum by host and A. campylopodum (Wilks’ l 5 0.0702, 16 Mathiasen, Kenaley, and Daugherty ALISO

Table 3. Morphological measurements for Arceuthobium divaricatum from Pinus edulis and from P. monophylla and P. californiarum subsp. fallax combined. Data are listed as mean, (SD), range, [n]. Means followed by different capital letters in the same row were significantly different using ANOVA (a 5 0.05). Lower case letters in brackets designate sample sizes already listed in the same column. Plant heights are in cm and all other measurements in mm.

Arceuthobium divaricatum on Pinus monophylla Character Arceuthobium divaricatum on Pinus edulis and P. californiarum subsp. fallax

Plant height Female 10.1 A (2.2) 5.2216.8 [300a] 11.8 B (2.9) 6.3222.7 [300a] Male 10.6 A (2.3) 5.7220.4 [a] 12.8 B (3.8) 5.9230.4 [a] Basal diameter Female 2.6 A (0.5) 1.725.0 [a] 2.6 A (0.5) 1.324.4 [a] Male 2.5 A (0.5) 1.624.9 [a] 2.4 A (0.5) 1.624.2 [a] Length of third internode Female 11.3 A (2.5) 5.8220.6 [a] 11.8 A (2.6) 6.9221.9 [a] Male 11.5 A (2.4) 5.5220.1 [a] 11.9 A (2.7) 6.0221.8 [a] Width of third internode Female 1.9 A (0.3) 1.323.0 [a] 1.9 A (0.3) 1.223.2 [a] Male 1.9 A (0.3) 1.223.0 [a] 1.9 A (0.3) 1.323.1 [a] Staminate spike length 9.5 A (3.3) 4.1231.8 [280b] 9.4 A (2.9) 3.2221.4 [a] Staminate spike width 1.7 A (0.1) 1.122.3 [b] 1.7 A (0.1) 1.322.0 [a] Flower diameter 3-merous 2.2 A (0.3) 1.423.1 [b] 2.2 A (0.3) 1.423.2 [a] Petal lobe length 1.1 A (0.2) 0.721.6 [b] 1.1 A (0.2) 0.721.6 [a] Petal lobe width 1.0 A (0.1) 0.621.4 [b] 1.0 A (0.1) 0.621.5 [a] Anther diameter 0.4 A (0.1) 0.320.7 [b] 0.4 A (0.1) 0.320.6 [a] Anther distance from tip 0.4 A (0.1) 0.220.7 [b] 0.4 A (0.2) 0.220.8 [a] Mean fruit length 4.4 A (0.4) 3.225.3 [290c] 4.4 A (0.3) 3.425.1 [a] Mean fruit width 2.6 A (0.3) 1.923.5 [c] 2.6 A (0.2) 1.923.2 [a] Seed length 2.1 A (0.2) 1.622.8 [c] 2.2 B (0.3) 1.623.1 [a] Seed width 1.1 A (0.1) 0.821.3 [c] 1.1 A (0.1) 0.821.3 [a]

Table 4. Comparison of morphological measurements for Arceuthobium divaricatum and A. campylopodum: A. divaricatum data is from Pinus monophylla and P. californiarum subsp. fallax combined, and P. edulis alone. Data are listed as mean, (SD) [n]. Means followed by different capital letters in the same row were significantly different using ANOVA followed by a Tukey’s Post Hoc HSD test (a 5 0.05). Lower case letters in brackets designate sample sizes already listed in the same column. Plant heights are in cm and all other measurements in mm.

Arceuthobium divaricatum Arceuthobium divaricatum from Pinus monophylla and Character from Pinus edulis P. californiarum subsp. fallax Arceuthobium campylopodum

Plant height Female 10.1 A (2.2) [300a] 11.8 B (2.9) [230a] 10.4 A (2.7) [600a] Male 10.6 A (2.3) [a] 12.8 B (3.8) [a] 9.7 C (3.0) [a] Basal diameter Female 2.6 A (0.5) [a] 2.6 A (0.5) [a] 3.4 B (0.7) [a] Male 2.5 A (0.5) [a] 2.4 A (0.5) [a] 3.2 B (0.6) [a] Length of third internode Female 11.3 A (2.5) [a] 11.8 B (2.6) [a] 13.0 B (3.1) [a] Male 11.5 A (2.4) [a] 11.9 A (2.7) [a] 11.9 A (3.3) [a] Width of third internode Female 1.9 A (0.3) [a] 1.9 A (0.3) [a] 2.5 B (0.4) [a] Male 1.9 A (0.3) [a] 1.9 A (0.3) [a] 2.5 B (0.4) [a] Staminate spike length 9.5 A (3.3) [280b] 9.4 A (2.9) [a] 12.7 B (4.8) [760b] Staminate spike width 1.7 A (0.1) [b] 1.7 A (0.1) [a] 3.0 B (0.3) [b] Flower diameter 3-merous 2.2 A (0.2) [b] 2.2 A (0.2) [a] 3.1 B (0.4) [400c] Petal lobe length 1.1 A (0.2) [b] 1.1 A (0.2) [a] 1.6 B (0.2) [c] Petal lobe width 1.0 A (0.1) [b] 1.0 A (0.2) [a] 1.4 B (0.2) [c] Anther diameter 0.4 A (0.1) [b] 0.4 A (0.1) [a] 0.6 B (0.1) [c] Anther distance from tip 0.4 A (0.1) [b] 0.4 B (0.1) [a] 0.6 C (0.1) [c] Mean fruit length 4.4 A (0.4) [290c] 4.4 A (0.3) [a] 5.4 B (0.5) [480d] Mean fruit width 2.6 A (0.3) [c] 2.6 B (0.2) [a] 3.7 B (0.4) [d] Seed length 2.1 A (0.2) [c] 2.2 B (0.3) [a] 3.5 C (0.4) [d] Seed width 1.1 A (0.1) [c] 1.1 A (0.1) [a] 1.5 B (0.2) [d] VOLUME 34(1) Morphometric Analysis of Dwarf Mistletoes 17

Table 5. Summary of the principal characters separating Female plants of A. divaricatum collected from P. edulis were Arceuthobium campylopodum and A. divaricatum.Datafor often misclassified to P. monophylla (18.6%,) and P. califor- morphological characters are means; plant heights in cm and all niarum subsp. fallax (12.4%). Likewise, A. divaricatum on P. other measurements in mm. Numbers in bold represent key morphological differences between the taxa. monophylla and P. californiarum subsp. fallax were consistent- ly and incorrectly classified $34.1% of the time as parasitizing Arceuthobium Arceuthobium P. edulis. Character campylopodum divaricatum A similar pattern in misclassification was also apparent for Plant height predicting host trees of A. divaricatum according to morpho- Female 10.4 10.9 logical characteristics of male plants, where the host tree was Male 9.7 11.7 predicted correctly only 72.9%, 57.1%, and 62.9% of the time Plant color Yellow, Olive-green, green, for male plants infecting P. edulis, P. monophylla, and P. yellow-brown dark brown californiarum subsp. fallax, respectively (Table 6). Although Basal diameter the precision in classification of A. divaricatum to its pinyon Female 3.4 2.6 host increased markedly following DFAs on a randomized Male 3.2 2.6 Width of third internode resample of female and male plants, the female plants of Female 2.5 1.9 A. divaricatum on P. monophylla and P. californiarum subsp. Male 2.5 1.9 fallax were, again, frequently misclassified and placed correct- Staminate spike width 3 1.7 ly to host only 68% of the time. Thus, morphological Flower diameter measurements for male and female plants on all pinyon hosts 3-merous 3.1 2.2 were combined (combined datasets) to further assess species 4-merous 4.2 3.1 membership between A. divaricatum and A. campylopodum as Petal length 1.6 1.1 well as identify the morphological characters contributing Petal width 1.4 1 Fruit length 5.4 4.4 most to interspecific separation. Fruit width 3.7 2.6 Means and associated 95% confidence intervals for mor- Seed length 3.5 2.2 phological characters of female and male plants across Seed width 1.5 1.1 predicted species according to full-model DFA are presented Host(s)a in Table 7. Discriminant function analysis using separately Principal Pinus jeffreyi; P. Pinus californiarum eight female and 10 male morphological characters (full- ponderosa var. subsp. fallax; P. model) clearly demonstrated separation of Arceuthobium ponderosa cembroides; P. campylopodum and A. divaricatum; $98.3% of female and discolor; P. edulis; P. monophylla; P. male plants identified via field diagnosis as A. divaricatum or quadrifolia A. campylopodum were predicted correctly to species (Table 8). Secondary P. attenuata; P. None For DFA of female plants, results indicated significant coulteri differences between multivariate means for A. campylopodum

Occasional P. contorta var. None and A. divaricatum (Wilks’ l 5 0.1353, Exact F8,1051 5 839.06, murrayana and var. P , 0.0001; Hotelling-Lawley Trace 5 6.3868, Exact F8,1051 5 latifolia; 839.06, P , 0.0001; Pillai’s Trace 5 0.8646, Exact F8,1051 5 P. sabiniana 839.06, P , 0.0001). The discriminant function accounted for Rare P. lambertiana None Immune P. monophylla P. jeffreyi; P. 100% of the total variation as only one dimension is possible ponderosa var. when assigning two groups. Female plants of Arceuthobium scopulorum divaricatum were correctly classified (predicted/actual) to species 98.3% (570/580) of the time, and hence the percentage a Host susceptibility classification based on information in Hawks- of female A. divaricatum assigned to A. campylopodum was worth and Wiens (1996) from their field observations only. only 1.7% (10/580). Similarly, 100% of female A. campylopo- dum were classified correctly when considering all eight female Approx. F30,3367.3 5 165.16, P , 0.0001; Pillai’s Trace 5 morphological characters. Examination of the standardized 1.0581, Approx. F30,3447 5 62.61, P , 0.0001). The first two correlation coefficients (scc) indicated that seed length (scc 5 discriminant functions (canonicals) for the DFA of female and 1.63), width of the third internode (scc 5 1.48), and fruit width male plants accounted for $98.3% and #1.3% of the total (scc 5 0.85) were most strongly correlated with the discrim- variation (Fig. 3), whereas the third discriminant function for inant function, thereby contributing most to defining species female and male plants accounted for #0.22 of the total membership for female plants. Using these three characters variation. Female and male plants of A. campylopodum were alone, female plants of A. campylopodum and A. divaricatum readily distinguished morphologically from A. divaricatum on were classified correctly to species 100% (480/480) and 97.9% all three pinyons because 100% (600/600) and 99.8% (479/480) (568/580) of the time, respectively. Moreover, with the addition of male and female plants diagnosed a priori as A. of plant height, fruit length, and length of the third internode campylopodum were predicted correctly (%, predicted/actual) as predictor variables, the percentage of female A. divaricatum to this species using 8 and 10 morphological characters, predicted correctly to species increased slightly to 98.6% (572/ respectively (Table 6). However, samples of female or male 580)—the highest correct classification percentage achieved for plants of A. divaricatum from the various pinyon hosts, female A. divaricatum by either the full-model or stepwise- analyzed as above, were indistinguishable (Table 6; Fig. 3). DFA (Table 8). 18 Mathiasen, Kenaley, and Daugherty ALISO

Table 6. Discriminant function analyses (DFAs) of female and male plants using complete records and resampled data for Arceuthobium campylopodum and, partitioned by pinyon host, A. divaricatum. Data are presented as: correct classification (count [N]), % [predicted/actual].

Arceuthobium divaricatum Arceuthobium Total (N) campylopodum Pinus edulis Pinus californiarum subsp. fallax Pinus monophylla

Complete – Female A. campylopodum 480 479, 99.8% 0, 0.0% 1, 0.2% 0, 0.0% A. divaricatum P. edulis 290 3, 1.0% 197, 67.9% 36, 12.4% 54, 18.6% P. californiarum subsp. fallax 70 0, 0.0% 29, 41.4% 32, 45.7% 9, 12.9% P. monophylla 220 2, 0.9% 75, 34.1% 35, 15.9% 108, 49.1% Resample – Female A. campylopodum 25 25, 100% 0, 0.0% 0, 0.0% 0, 0.0% A. divaricatum P. edulis 25 0, 0.0% 23, 92.0% 0, 0.0% 2, 8.0% P. californiarum subsp. fallax 25 0, 0.0% 7, 28.0% 17, 68.0% 1, 4.0% P. monophylla 25 0, 0.0% 4, 16.0% 4, 16.0% 17, 68.0% Complete – Male A. campylopodum 600 600, 100% 0, 0.0% 0, 0.0% 0, 0.0% A. divaricatum P. edulis 280 1, 0.4% 204, 72.9% 44, 15.7% 31, 11.1% P. californiarum subsp. fallax 70 0, 0.0% 16, 22.9% 44, 62.9% 10, 14.3% P. monophylla 210 0, 0.0% 46, 21.9% 44, 21.0% 120, 57.1% Resample – Male A. campylopodum 25 25, 100% 0, 0.0% 0, 0.0% 0, 0.0% A. divaricatum P. edulis 25 0, 0.0% 23, 92.0% 2, 8.0% 0, 0.0% P. californiarum subsp. fallax 25 0, 0.0% 2, 8.0% 21, 84.0% 2, 8.0% P. monophylla 25 0, 0.0% 4, 16.0% 2, 8.0% 22, 88.0%

Significant differences among 10 male morphological char- acters of Arceuthobium campylopodum and A. divaricatum were Table 7. Discriminant function analyses (DFA) of male and also revealed by DFA (Wilks’ l 5 0.0824, Exact F10,1149 5 female plants: comparison of morphological characters by predicted 1279.59, P, 0.0001; Hotelling-Lawley Trace 5 11.1366, Exact classification to A. campylopodum and A. divaricatum using complete F10,1149 5 1279.59, P, 0.0001; Pillai’s Trace 5 0.9176, data per taxon. Ninety-five percent confidence intervals (6) were Exact F10,1149 5 1279.59, P, 0.0001), classifying correctly computed for comparison of mean differences. Combined analysis of 100% (600/600) and 99.8% (559/560) of male plants identified plants on Pinus edulis, P. monophylla,andP. californiarum a priori to A. campylopodum and A. divaricatum, respectively. subsp. fallax. Staminate spike width (scc 5 0.97) was most strongly correlated to the discriminant function and, using this Comparison between species (predicted) morphological character alone (i.e., minus all other male plant Arceuthobium Arceuthobium Character by plant sex campylopodum divaricatum measurements), resulted in the total correct classification to species of 99.8% (1158/1160) across all male plants—100% Female (600/600) A. campylopodum and 99.6% (558/560) A. divaricatum Plant height (PH) 10.3 (60.24) 10.9 (60.22) (Table 8). Therefore, although univariate statistics pre- Basal diameter (BA) 3.4 (60.06) 2.6 (60.04) sented herein demonstrated significant differences between A. Length of third internode (LTI) 13.1 (60.27) 11.5 (60.21) campylopodum and A. divaricatum across 9 of 10 male Width of third internode (WTI) 2.5 (60.03) 1.9 (60.02) Fruit length (FL) 5.4 (60.04) 4.4 (60.03) characters used in the full-model DFA, utilizing the other Fruit width (FW) 3.7 (60.04) 2.6 (60.02) eight male characters (e.g., plant height, basal diameter, Seed length (SL) 3.5 (60.04) 2.2 (60.02) staminate spike length, anther measurements, etc.) within the Seed width (SW) 1.5 (60.02) 1.1 (60.01) DFA model were unnecessary to delimit male plants to species Male since DFA using only staminate spike width correctly Plant height (PH) 9.7 (60.24) 11.7 (60.28) predicted species membership for all but two male plants of Basal diameter (BA) 3.2 (60.05) 2.5 (60.04) A. divaricatum (Table 8). Length of third internode (LTI) 11.9 (60.26) 11.7 (60.21) Width of third internode (WTI) 2.5 (60.03) 1.9 (60.02) Petal length (PL) 1.5 (60.02) 1.1 (60.01) DISCUSSION Petal width (PW) 1.4 (60.02) 1.0 (60.01) Classifying Arceuthobium campylopodum and A. divarica- Anther diameter (AD) 0.6 (60.01) 0.4 (60.01) tum as conspecific or the latter species as a subspecies of A. Anther distance from tip (ADT) 0.6 (60.01) 0.4 (60.01) campylopodum is not supported by our analyses of the Staminate spike length (SSL) 12.9 (60.40) 9.5 (60.26) Staminate spike width (SSW) 3.0 (60.02) 1.7 (60.01) morphological characters for these taxa as both species can VOLUME 34(1) Morphometric Analysis of Dwarf Mistletoes 19

Table 8. Forward-stepwise discriminant function analysis (DFA) for female and male plants of Arceuthobium campylopodum and A. divaricatum: correct classification counts (N 5 predicted/actual) and percentages (%, predicted/actual) with the sequential addition of morphological characters (steps) most-to-least correlated to the discriminant function. Anther diameter (AD); anther distance to tip (ADT); basal diameter (BD); fruit length (FL); fruit width (FW); length of third internode (LTI); plant height (PH); petal length (PL); petal width (PW); seed length (SL); staminate spike length (SSL); staminate spike width (SSW); seed width (SW); and width of the third internode (WTI).

Correct classification to species Arceuthobium campylopodum Arceuthobium divaricatuma Stepwise DFA by plant sex (step: morphological character[s]) Total (%) Count % Count %

Female 1: SL 95.5 451/480 94.0 561/580 96.7 2: SL, FW 98.4 478/480 99.6 565/580 97.4 3: SL, FW, WTI 98.9 480/480 100.0 568/580 97.9 4: SL, FW, WTI, PH 99.0 479/480 99.8 570/580 98.3 5: SL, FW, WTI, PH, FL 99.1 479/480 99.8 571/580 98.4 6: SL, FW, WTI, PH, FL, LTI 99.2 480/480 100.0 572/580 98.6 7: SL, FW, WTI, PH, FL, LTI, BD 99.2 480/480 100.0 571/580 98.4 8 (Full-model): SL, FW, WTI, PH, FL, LTI, BD, SW 99.1 480/480 100.0 570/580 98.3 Male 1: SSW, 99.8 600/600 100.0 558/560 99.6 2: SSW, WTI 99.9 600/600 100.0 559/560 99.8 3: SSW, WTI, SSL 99.9 600/600 100.0 559/560 99.8 4: SSW, WTI, SSL, PH 99.9 600/600 100.0 559/560 99.8 5: SSW, WTI, SSL, PH, LTI 99.9 600/600 100.0 559/560 99.8 6: SSW, WTI, SSL, PH, LTI, PW 99.9 600/600 100.0 559/560 99.8 7: SSW, WTI, SSL, PH, LTI, PW, BD 99.9 600/600 100.0 559/560 99.8 8: SSW, WTI, SSL, PH, LTI, PW, BD, ADT 99.9 600/600 100.0 559/560 99.8 9: SSW, WTI, SSL, PH, LTI, PW, BD, ADT, AD 99.9 600/600 100.0 559/560 99.8 10 (Full-model): SSW, WTI, SSL, PH, LTI, PW, BD, ADT, AD, PL 99.9 600/600 100.0 559/560 99.8

a Combined data for plants collected on Pinus edulis, P. monophylla, and P. californiarum subsp. fallax. be identified easily by differences in one or more morpholog- infection by A. divaricatum (Hawksworth and Wiens 1996). ical characters as well as their host affinities (Tables 5, 7, and It is probable that P. ponderosa is also immune to infection 8; Fig. 3). For example, the basal diameter, width of the third by A. divaricatum: Hawksworth and Wiens (1996) observed internode, and the width of mature staminate spikes were that where P. ponderosa was sympatric with infected significantly smaller for plants of A. divaricatum than those of pinyons, A. divaricatum was found infecting only pinyons. A. campylopodum; overall, male and female plants of Although they refrained from classifying P. ponderosa A. divaricatum were much more slender when compared to as immune to A. divaricatum,HawksworthandWiens plants of A. campylopodum. This characteristic is easily did suggest P. ponderosa var. scopulorum Engelm. (Rocky discernible when male or female plants of A. divaricatum Mountain ponderosa pine) may be immune to A. divarica- and A. campylopodum are placed side-by-side for visual tum in the Southwest. comparisons (Fig. 4–5). However, plant size alone does not Because the dwarf mistletoes are extremely important both easily distinguish these species even though the mean heights ecologically and economically, emphasis must be placed on of male and female plants were significantly different their ecological and pathological roles in forest ecosystems. (Table 1). Other characters that distinguished A. divaricatum These two dwarf mistletoes clearly have very different host from A. campylopodum were its much smaller 3-merous ranges and hence, their pathological significance in forests flowers, rare formation of 4-merous flowers, and smaller of the western United States is also distinct. Any efforts to fruits and seeds (Tables 7 and 8). Furthermore, although manage populations of these parasites to mitigate their effects plant color is a qualitative character, A. divaricatum can on tree growth and mortality within severely infested stands usually be distinguished using plant color as its female plants must consider these differences (Hawksworth and Wiens are typically brown or green-brown while those of A. 1996). Recognition of the host affinities developed by dwarf campylopodum typically are yellow, yellow-brown, or light mistletoes is critical in their classification because we consider brown (Fig. 4–5). Again, these color differences can be easily differences in host preference(s) to reflect corresponding and observed when recently collected plants are compared side-by- underlying genetic and physiological differentiation among side; upon drying these color differences are more difficult to dwarf mistletoes. observe. In addition to our morphological analyses, the separation A further difference between these dwarf mistletoes is of Arceuthobium divaricatum from A. campylopodum is their host range; the principal and only hosts of Arceutho- also supported by isozyme and molecular studies. Nickrent bium divaricatum are pinyons (Table 5). In contrast, A. (1986, 1996) reported that, based on isozyme analyses, A. campylopodum primarily parasitizes Pinus ponderosa and P. divaricatum was most closely aligned with and biochemi- jeffreyi with the latter species claimed to be immune to cally similar to A. douglasii Engelm. (Douglas-fir dwarf 20 Mathiasen, Kenaley, and Daugherty ALISO mistletoe), and was clearly distinct from A. campylopodum. recommend these populations be considered for separate Both A. divaricatum and A. douglasii shared 16 alleles across taxonomic recognition as subspecies of A. divaricatum.The 6 loci and both species were fixed for one isozyme host-parasite and/or environmental factors contributing to the (glutamate dehydrogenase GDH66), which was absent in significant differences between mean plant heights for A. all other species of Arceuthobium studied, including A. divaricatum collected from P. edulis and P. monophylla/fallax campylopodum. This demonstrated that A. divaricatum was remain unclear. genetically distinct from A. campylopodum.Furthermore, We have not reported morphological measurements for Nickrent et al. (2004) presented phylogenetic analyses using plants of Arceuthobium divaricatum collected from Pinus sequence data of ITS and chloroplast trn-L DNA that californiarum subsp. californiarum, P. discolor, P. cem- also demonstrated A. divaricatum was most closely related broides,orP. quadrifolia, but we did observe infection on phylogenetically to A. douglasii,notA. campylopodum. the first three hosts and we measured a few male and female Based on DNA sequence data, Nickrent et al. (2004) placed plants collected from P. discolor in New Mexico and P. A. divaricatum in a clade with A. douglasii in section Minuta cembroides in Texas. Plant, flower, fruit, and seed dimen- Hawksw.&WiensratherthansectionCampylopoda sions measured from P. discolor were morphologically Hawksw. & Wiens, where A. campylopodum is presently similar to those of plants collected from P. edulis.Plant classified. Most recently, Reif et al. (2015) also demonstrat- dimensions measured from P. cembroides were similar to ed that A. divaricatum was genetically different from three plants from P. edulis as well. As reported by Hawksworth species in section Campylopoda (A. apachecum Hawksw. & and Wiens (1996), infection of P. californiarum subsp. Wiens, A. blumeri A. Nelson, and A. cyanocarpum (A. californiarum, P. cembroides,andP. discolor was severe Nelson ex Rydberg) Coulter & Nelson) using amplified where we observed these hosts and we agree that these fragment length polymorphism (AFLP) analyses. Although pinyons should also be classified as principal hosts of A. A. campylopodum was not included in their study of dwarf divaricatum, even though they are not common within its mistletoes of section Campylopoda that parasitize white geographic range. Although A. divaricatum has been pines (Pinus subgenus Strobus), A. divaricatum and A. reported parasitizing P. quadrifolia on the east slope of the douglasii were utilized as outgroup taxa and were genetically Laguna Mountains (San Diego County, CA; Beauchamp distinct to the aforementioned white pine dwarf mistletoes 1986), we did not observe infection of this host in the presently classified to section Campylopoda. Therefore, the Laguna Mountains where we searched for this host/dwarf circumscription of A. divaricatum under A. campylopodum mistletoe combination in 2015. While A. divaricatum has (Baldwin et al. 2012) is not justified based on our rigorous been collected on P. quadrifolia from the Sierra Jua´rez and morphological analyses, their apparent host affinities based reported in the Sierra San Pedro Ma´rtir, Baja California, we on field observations, and the isozyme and molecular studies were unable to visit these mountains during this study. reportedtodate. While Hawksworth and Wiens (1972, 1996) reported the It is interesting to note that male and female mean plant maximum height of plants of Arceuthobium divaricatum as heights of Arceuthobium divaricatum collected from Pinus 13 cm, we measured female plants greater than 20 cm and monophylla and P. californiarum subsp. fallax were signifi- male plants greater than 30 cm in height (Table 1). We cantly larger on average than those of plants collected from found that fruits averaged 4.5 mm in length and 2.6 mm in P. edulis (Tables 2 and 3). The differences in plant heights width whereas Hawksworth and Wiens reported fruits were may be related to the greater amount of annual precipitation only 3.5 3 2.0 mm. Furthermore, Hawksworth and Wiens at sites typically colonized by P. monophylla—primarily did not report that A. divaricatum occasionally formed 4- during the late fall, winter, and early spring—compared to merous flowers, only 3-merous, and they reported that that of P. edulis (Malusa 1992; Cole et al. 2008). Most flowers were larger (2.5 mm) in diameter than the 3-merous notably, P. monophylla typically receives more rain on average flowers we measured (mean 5 2.2 mm). Possible reasons for in May and June (45 mm) than P. edulis (34 mm) (Cole et al. these differences were that we measured many more 2008). Therefore, the larger plant heights of A. divaricatum collections (60) than Hawksworth and Wiens (19) and our sampled from P. monophylla may have been affected directly by the increased moisture present where this host occurs or measurements were made on fresh material, not dried indirectly by greater vigor of this host produced by the higher herbarium specimens. The discrepancy between our di- moisture regimes it grows under, particularly during the ameter measurements for 3-merous flowers, however, is spring months of May and June. However, plant heights of A. unclear, unless they reported the maximum diameter they divaricatum collected from P. californiarum subsp. fallax were observed for flowers. We measured 3-merous flowers as also larger than those collected from P. edulis and these large as 3.2 mm and 4-merous flowers as large as 4.1 mm. pinyons grow under similar bi-seasonal precipitation patterns Although we don’t know the reasons for these differences, it where they receive most of their precipitation during the is evident from our morphological measurements that summer (July to September) monsoon season (Malusa 1992; plants, flowers, and fruits of A. divaricatum are often much Cole et al. 2008). Therefore, precipitation patterns experi- larger than what has been reported previously in the enced by the pinyon hosts of A. divaricatum alone probably literature. don’t account for the greater mean heights of plants collected In light of the morphological data presented herein, previous from P. monophylla and P. californiarum subsp. fallax. isozyme and molecular findings, and the major discontinuities Because the mean plant height was the only significantly in the host affinities for each taxon, we advocate retention of different morphological character between the populations of separate species status for Arceuthobium campylopodum and A. divaricatum on P. monophylla and P. edulis,wedon’t A. divaricatum. VOLUME 34(1) Morphometric Analysis of Dwarf Mistletoes 21

KEY FOR IDENTIFICATION OF ARCEUTHOBIUM CAMPYLOPODUM [eds.], Mistletoes of North American conifers. General Technical AND A. DIVARICATUM Report RMRS-GTR-98, USDA Forest Service, Rocky Mountain Research Station, Ogden, UT. 1. Basal diameters usually .3 mm; width of third internode MALUSA, J. 1992. Phylogeny and biogeography of the pinyon pines usually .2 mm; staminate spike width usually .2mm; (Pinus subsect. Cembroides). Syst. Bot. 17: 42–66. staminate flowers 3- and 4-merous and .3 mm across; MATHIASEN,R.L.AND C. M. DAUGHERTY. 2007. Arceuthobium mature fruits typically .5 mm long and 3 mm wide; seeds tsugense subsp. amabilae, a new subspecies of hemlock dwarf about 3.5 31.5mm; primarilyparasitic onPinus ponderosa mistletoe (Viscaceae) from Oregon. Novon 17: 222–227. and P. jeffreyi; also common on P. attenuata and P. AND .2009a. Arceuthobium abietinum subsp. wiensii, coulteri ...... Arceuthobium campylopodum a new subspecies of fir dwarf mistletoe (Viscaceae) from northern California and southern Oregon. Madron˜o 56: 118–126. 1’. Basal diameters usually ,3 mm; width of third AND . 2009b. Additional morphological measurements internode usually ,2 mm; staminate spike width usually of Arceuthobium siskiyouense and A. monticola (Viscaceae). J. Bot. ,2 mm; staminate flowers predominantly 3-merous and Res. Inst. Texas 3: 741–749. ,3 mm across; mature fruits ,5 mm long and 3 mm AND . 2013. Additional morphological measurements wide; seeds about 2.2 3 1.1 mm; parasitic on pinyon of Arceuthobium littorum and A. occidentale (Viscaceae). Phytologia pines ...... Arceuthobium divaricatum 95: 70–78. AND S. C. KENALEY. 2015a. A morphometric analysis of the ACKNOWLEDGMENTS dwarf mistletoes in the Arceuthobium campylopodum-occidentale complex (Viscaceae). Madron˜o 62: 1–20. The authors wish to express their appreciation to Tony AND . 2015b. A morphometric analysis of Arceutho- DeLuz for preparation of the maps used as Figures 1 and 2. bium campylopodum, A. laricis,andA. tsugense (Viscaceae). Phytologia 97: 200–218. NICKRENT, D. L. 1986. Genetic polymorphism in the morphologically LITERATURE CITED reduced dwarf mistletoes (Arceuthobium, Viscaceae): an electropho-

BALDWIN, B. G., D. GOLDMAN,D.J.KEIL,R.PATTERSON,T.J. retic study. Amer. J. Bot. 73: 1492–1502. ROSATTI, AND D. H. WILKEN. (Editors). 2012. The Jepson manual: . 1996. Molecular systematics, pp. 155–172. In F. G. Hawks- vascular plants of California, 2nd edition. University of California worth and D. Wiens, Dwarf mistletoes: biology, pathology, and Press, Berkeley. 1600 p. systematics. Agriculture Handbook 709, USDA Forest Service, BEAUCHAMP, R. M. 1986. A flora of San Diego County, California. Washington, DC. 410 p. Sweetwater Press, National City, CA. 241 p. . 2012. Justification for subspecies in Arceuthobium campy- COLE, K. L., J. FISHER,S.T.ARUNDEL,J.CANNELLA, AND S. SWIFT. lopodum (Viscaceae). Phytoneuron 51: 1–11. 2008. Geographical and climatic limits of needle types of one- and ,M.A.GARCI´A,M.P.MARTI´N, AND R. L. MATHIASEN. 2004. two-needled pinyon pines. J. Biogeogr. 35: 257–269. A phylogeny of all species of Arceuthobium (Viscaceae) using nuclear ENGELMANN, G. 1878. Description of Arceuthobium divaricatum in U.S. and chloroplast DNA sequences. Amer. J. Bot. 91: 125–138. th Geographical Survey West of the 100 Meridian (Wheeler Report) ,K.P.SCHUETTE, AND E. M. STARR. 1994. A molecular 6: 253. phylogeny of Arceuthobium based upon rDNA internal transcribed GILL, L. S. 1935. Arceuthobium in the United States. Trans. Conn. Acad. spacer sequences. Amer. J. Bot. 81: 1149–1160. Arts Sci. 32: 111–245. QUINN,G.P.AND M. J. KEOUGH. 2002. Experimental design HAWKSWORTH,F.G.AND D. WIENS. 1972. Biology and classification of and data analysis for biologists. Cambridge University Press, UK. dwarf mistletoes (Arceuthobium). Agriculture Handbook 401, 537 p. USDA Forest Service, Washington, DC. 234 p. REIF, B. P., R. L. MATHIASEN,S.C.KENALEY, AND G. J. ALLAN. 2015. AND . 1996. Dwarf mistletoes: biology, pathology, and Genetic structure and morphological differentiation of three western systematics. Agriculture Handbook 709, USDA Forest Service, North America dwarf mistletoes (Arceuthobium: Viscaceae). Syst. Washington, DC. 410 p. Bot. 40: 191–207. ,D.WIENS, AND B. W. GEILS. 2002. Arceuthobium in North ZIMMERMAN, D. W. 2004. A note on preliminary tests of equality of America, pp. 29–56. In B. W. Geils, J. Cibrian-Tovar, and B. Moody variances. Br. J. Math. Stat. Pychol. 57: 173–181. 22 Mathiasen, Kenaley, and Daugherty ALISO

Appendix 1. Collection locations and voucher specimen numbers for Arceuthobium campylopodum. Collections by R. L. Mathiasen (RLM). All vouchers were deposited at the University of Arizona Herbarium, Tucson (ARIZ), except for RLM 0938 (population 46) which was deposited at the Deaver Herbarium, Northern Arizona University, Flagstaff (ASC). Population numbers correspond to locations in Fig. 1.

Population Location Specimen number

1 4.5 km N of Gifford on St. Rte. 25 RLM 1202 2 20 km S of Fruitland on St. Rte. 25 RLM 1204 3 2 km NW of Nespelem on St. Rte. 155 RLM 1205 4 2.3 km N of Coeur d’Alene, ID on Fernan Lake Rd. RLM 1195 5 16 km S of Spokane on St. Rte. 195 RLM 1194 6 2.5 km W of St. Rte. 153 on Squaw Creek Rd. RLM 1208 7 Lake Wenatchee on Chiwawa River Loop Rd. RLM 1224 8 2.6 km W of Squilchuck St. Park on Ski Area Rd. RLM 1209 9 0.8 km W of St. Rte. 97 on St. Rte 970 RLM 1212 10 17.6 km E of White Pass on St. Rte. 12 RLM 1219 11 2 km N of Satus Pass on St. Rte. 97 RLM 1213 12 3 km S of Trout Lake on St. Rte. 141 RLM 1217 13 6.4 km W of Friend on Forest Rd. 27 RLM 1214 14 6.4 km S of Joseph on E shore of Wallowa Lake RLM 1191 15 9.4 km on Sheep Cr. Rd from Forest Rd. 51 RLM 1188 16 1.8 km E of Ochoco Summit on St. Rte. 26 RLM 1178 17 12.2 km W of St. Rte. 97 on St. Rte. 138 RLM 1171 18 15.2 km S of Sisters on Forest Rd. 16 RLM 1175 19 1 km from Forest Rd. 44 on Forest Rd. 4410 RLM 1173 20 Fort Klamath Cemetery on St. Rte. 62 RLM 1126 21 3 km W of Quartz Mtn. Pass on St. Rte. 140 RLM 1127 22 Warner Mtn. Ski Hill on St. Rte. 26 RLM 1130 23 3.4 km W of County Rd. 48 on Forest Rd. 73 RLM 1131 24 16 km N of Adin on St. Rte. 299/139 RLM 1132 25 6 km S of Takilma on Grayback Rd. RLM 1167 26 1 km S of Forest Rd. 17N26 on Forest Rd. 17N11 RLM 1166 27 6.2 km W of St. Rte. 96 on Dillon Mtn. Rd. RLM 1165 28 9.6 km S of Callahan on St. Rte. 3 RLM 1121 29 10 km E of St. Rte 3 on Forest Rd. 17 RLM 1120 30 2.4 km W of Stewart Hot Springs on Forest Rd. 17 RLM 1160 31 2 km N of St. Rte. 89 on Mt. Shasta Ski Park Rd. RLM 1158 32 0.1 km S of St. Rte. 299 on St. Rte. 89 RLM 1157 33 2 km S of Old Station on St. Rte. 44 RLM 1154 34 2 km W of St. Rte. 44 on Forest Rd. 101 RLM 1153 35 14.4 km W of Susanville on St. Rte. 36 RLM 1032 36 19.5 km N of Upper Lake on Pillsbury Lake Rd. RLM 0920 37 7.7 km N of Pollock Pines on Forest Rd. 4 RLM 1242 38 Entrance to Sugar Pine State Park RLM 1147 39 Bowers Mansion St. Park RLM 1146 40 1 km N of Markleeville on St. Rte. 89 RLM 1133 41 Silver Creek Campground on St. Rte. 4 RLM 1134 42 Column of the Giants on St. Rte. 108 RLM 1145 43 Pinecrest Transfer Station; 0.5 km W of Pinecrest RLM 1143 44 1 km W of Long Barn on St. Rte. 108 RLM 1142 45 8.5 km E of Crane Flat on St. Rte. 120 RLM 1138 46 2 km W of Big Creek on Shaver Lake Rd. RLM 0938 47 4.1 km W of Ranger Station on Kyle Canyon Rd. RLM 1137 48 8.5 km W of Sherman Pass on Forest Rd. 22S05 RLM 1296 49 2.2 km S of Troy Mdws. Campground RLM 1135 50 5.8 km N of Johnsonville Jct. on W. Divide Highway RLM 0985 51 Pine Flat, Sequoia Nat. For. RLM 0980 52 Tiger Flat, Sequoia Nat. For. RLM 0976 53 6.2 km S of St. Rte. 33 on Mt. Reyes Rd. RLM 1292 54 1.4 km W of Cloud Burst on St. Rte. 2 RLM 1304 55 1 km W of Big Pines on St. Rte. 2 RLM 1305 56 2.4 km N of Fawnskin on Forest Rd. 2N71 RLM 0986 57 1.9 km from St. Rte. 38 on Jenks Lake Rd. RLM 0973 58 Forest Service Ranger Station in Idyllwild RLM 0969 59 1.1 km S of the San Jacinto River on St. Rte. 74 RLM 0967 60 0.5 km S of Horse Heaven Campground RLM 1306 VOLUME 34(1) Morphometric Analysis of Dwarf Mistletoes 23

Appendix 2. Collection locations and voucher specimen numbers for Arceuthobium divaricatum. Collections by R. L. Mathiasen (RLM); all vouchers are deposited at the Herbarium of the Rancho Santa Ana Botanical Garden (RSA). Population numbers correspond to locations in Fig. 2.

Population Location Specimen number

1 3.8 km W of St. Rte. 395 on Leviathan Mine Rd. RLM 1390 2 6 km W of St. Rte. 395 on St. Rte. 89 RLM 1389 3 1.7 km S of Camp Antelope Rd. on Burcham Flat Rd. RLM 1377 4 3.2 km S of Benton Crossing on Chidago Cyn. Rd. RLM 1378 5 4.5 km S of St. Rte. 6 on Forest Rd. 1N14 RLM 1324 & 1341 6 4.8 km W of Carroll Summit on St. Rte. 772 RLM 1391 7 0.6 km W of Big Creek Campground S of Austin RLM 1392 8 15 km E of Austin on US 50 RLM 1393 9 Pole Creek Trailhead in Great Basin Nat. Park RLM 1394 10 1.8 km N of St. Rte. 168 on Bristlecone Pine Rd. RLM 1340 11 10.4 km S of Trona Jct. in Wildrose Cyn. RLM 1325 & 1339 12 1.6 km from Nat. For. on Horseshoe Mdw. Rd. RLM 1388 13 1.6 km W of Kern River on Sherman Pass Rd. RLM 1379 14 11.2 km W of Chimney Peak Rd. on Long Valley Rd. RLM 1380 15 0.3 km N of Lamont Peak on Chimney Peak Byway RLM 1381 16 4.8 km W of Kelso Valley Rd. on Piute Mtn. Rd. RLM 1387 17 2.2 km N of Lockwood Valley Rd. on Dome Sprs. Rd. RLM 1386 18 22 km W of Cuddy Valley Rd. on Lockwood Valley Rd. RLM 1385 19 2.7 km S of Lockwood Valley Rd. on Frazier Mtn. Rd. RLM 1384 20 3.2 km N of Big Pines Rd. on Mescal Cyn. Rd. RLM 1383 21 14.4 km S of Lucerne Valley on St. Rte. 18 RLM 1336 22 5.4 km S of St. Rte. 18 on Forest Rd. 3N03 RLM 1382 23 18.4 km W of St. Rte. 95 on Lee Cyn. Rd. RLM 1326 & 1338 24 Entrance to Caruthers Cyn., New York Mtns. RLM 1400 25 Entrance to Keystone Cyn., New York Mtns. RLM 1337 & 1371 26 0.5 km W of Hualapai Mtn. Rd. on DW Ranch Rd. RLM 1335 27 Little Wolf Hole Pass, Wolf Hole Mtns. RLM 1353 28 Anvil Rock Rd. exit on I-40 RLM 1397 29 17.6 km S of St. Rte. 260 on Fossil Creek Rd. RLM 1401 30 1 km S of Bell Rock on St. Rte 179 RLM 1365 31 N slope of Sugarloaf Mtn., Sedona RLM 1349 & 1355 32 1.6 km N of St. Rte. 260 on Forest Rd. 504 west of Heber RLM 1334 & 1356 33 3.2 km S of Eager on Forest Rd. 285 RLM 1327 & 1357 34 1 km N of Walnut Cyn. Nat. Monument RLM 1342 35 4.8 km E of Grand Cyn. Nat. Park on St. Rte. 64 RLM 1348 36 1.6 km E of Grand Cyn. Caverns on St. Rte. 66 RLM 1399 37 11.2 km E of Mt. Trumbull Schoolhouse on Loop Rd. RLM 1354 38 North Timp Pt. on North Rim Grand Cyn. RLM 1314 & 1316 39 12.1 km E of Jacob Lake on St. Rte. 89A RLM 1315 40 4.2 km S of Mt. Carmel Jct. on St. Rte. 89 RLM 1350 41 1.6 km W of Tropic on Bryce Way RLM 1351 42 5 km S of Boulder on St. Rte. 12 RLM 1352 43 0.3 km N of Forest Rd. 2653 on Forest Rd. 022 RLM 1396 44 1.6 km E of Devil’s Cyn. viewpoint on I-70 RLM 1395 45 6 km W of St. Rte. 275 on St. Rte. 95 RLM 1398 46 1.6 km E of La Sal on St. Rte. 46 RLM 1346 47 Colorado Nat. Monument. North Campground RLM 1366 48 6.4 km N of US 50 on St. Rte. 347 RLM 1367 49 19 km E of St. Rte. 145 on St. Rte. 90 RLM 1368 50 12.8 km W of Bedrock on St. Rte. 90 RLM 1369 51 3.2 km S of St. Rte. 164 on County Rd. 26 RLM 1345 52 17.6 km S of the Colorado St. boundary on St. Rte. 511 RLM 1344 53 0.4 km W of Coolidge on I-40 RLM 1343 54 14.4 km N of St. Rte. 60 on Priest Cyn. Rd. RLM 1330 & 1360 55 12.8 km W of Magdalena on St. Rte. 60 RLM 1329 & 1359 56 3.2 km W of Pie Town on St. Rte. 60 RLM 1328 & 1358 57 9.6 km E of St. Rte. 180 on Mogollon Rd. RLM 1333 58 6.4 km N of Fort Bayard on Old Wagon Rd. RLM 1363 59 1.6 km S of Nogal on St. Rte 37 RLM 1331 & 1361 60 0.3 km E of Forest Rd. 163 above La Luz Cyn. RLM 1332 & 1362